Prion diseases, otherwise known as Transmissible Spongiform Encephalopathies (TSEs), are a family of progressive neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce classic immune response. The causative agent of TSEs is believed to be a prion. A prion is a transmissible agent mostly consisting of a conformationally changed prion protein that is able to induce abnormal folding of normal cellular prion protein. Prion replication in the brain leads to brain damage and the characteristic signs and symptoms of the disease. Human prion diseases are rare, usually rapidly progressive and fatal; no preclinical diagnostic test or treatment is currently available.
The normal cellular prion protein is found in various organs and tissues throughout the body, including the brain, in healthy people and animals. However, prion protein found in the brains of disease-affected people or animals has a different “mis-folded” structure and is partially resistant to proteases. The normal cellular form of the prion protein is generally called PrPC (the “c” refers to “cellular”). The infectious form is variously called PrPSc (the “Sc” is from “scrapie”); PrPSc, TSE, CJD, GSS, FFI, BSE, CWD, etc (the Sc, TSE, CJD, GSS, FFI, and CWD refer to the abnormal protein of a TSE disease, and more specifically to scrapie, various forms of Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, bovine spongiform encephalopathy, chronic wasting disease, etc.); and more generally PrPd (the “d” refers to “disease-associated” prion protein).
Human PrPC is a 253-amino acid protein produced from a single-copy gene located on chromosome 20 (LIAO et al. “Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications,” Science, 1986, Vol. 233, pp. 364-367) which undergoes various posttranslational modifications that include formation of a disulphide bond (STAHL et al., “Prions and prion proteins,” FASEB J., 1991, Vol. 5, pp. 2799-2807), glycosylation (RUDD et al., “Prion glycoprotein: structure, dynamics, and roles for the sugars,” Biochemistry, 2001, Vol. 40, pp. 3759-66), removal of 22 amino acids from C-terminus and addition of glycophosphotidylinositol (GPI) moiety (STAHL et al., “; Cell, 1987, Vol. 51, pp. 229-40). The normal cellular protein is attached to the plasma membrane through a GPI anchor and has a predominantly alpha helical structure. The fully glycosylated protein has a molecular weight of 38 kDa, is monomeric in structure and sensitive to proteolysis (CAUGHEY et al., “Prions and their partners in crime,” Nature, 2006, Vol. 443, pp. 803-10, Review). PrPC is known to interact with various proteins, including heat-shock proteins (EDENHOFER et al., “Prion protein PrPc interacts with molecular chaperones of the Hsp60 family,” J Virol., 1996, Vol. 70, pp. 4724-8), a 37 kDa/67 kDa laminin receptor (RIEGER et al., “The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells,” Nat Med., 1997, Vol. 3, pp. 1383-8; GAUCZYNSKI et al., “The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein,” EMBO J., 2001, Vol. 20, pp. 5863-75), stress-inducible protein-1 of 66 kDa predominantly present in cytoplasm (ZANATA et al., “Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection,” EMBO J., 2002, Vol. 21, pp. 3307-16), plasminogen (ELLIS et al., “Plasminogen activation is stimulated by prion protein and regulated in a copper-dependent manner,” Biochemistry, 2002, Vol. 41, pp. 6891-6; PRAUS et al., “Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH2-terminal fragment PrP23-110,” Thromb Haemost., 2003, Vol. 89, pp. 812-9; KORNBLATT et al., “The fate of the prion protein in the prion/plasminogen complex,” Biochem Biophys Res Commun., 2003, Vol. 305, pp. 518-22), a neuronal cell adhesion molecule (NCAM) (SANTUCCIONE et al., “Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth,” J Cell Biol., 2005, Vol. 169, pp. 341-54), heparan sulphate proteoglycans (HPSG) (HORONCHIK et al., “Heparan sulfate is a cellular receptor for purified infectious prions,” J Biol Chem., 2005, Vol. 280, p. 17062, Epub., 2005 Jan. 24), the low-density lipoprotein receptor-related protein (LRP1) (TAYLOR et al., “Role of lipid rafts in the processing of the pathogenic prion and Alzheimer's amyloid-beta proteins,” Semin Cell Dev Biol., 2007, Vol. 18, pp. 638-48, Epub., 2007 Jul. 24, Review; PARKYN et al., “LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein,” J Cell Sci., 2008, Vol. 121(Pt 6), pp. 773-83, Epub., 2008 Feb. 19; Cervenakova et al., unpublished data, 2004), other LDL receptor superfamily members, megalin receptor and VLDLR (Cervenakova et al., unpublished data, 2004). It has been suggested that PrPC could function as a part of the LRP1 scavenger complex, because its N-terminal domain has multiple binding motifs (CAUGHEY et al., “Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: A common mechanism for anti-TSE compounds?” Acc Chem Res., 2006, Vol. 39, pp. 646-53) and its hydrophobic sequence (amino acids 112-130) exposed to an aqueous environment could bind to denatured proteins as PrPC rapidly traffics across the neuronal surface. Recently, it has been shown that LRP1 binds to and is involved in both the biosynthetic and the endocytic trafficking of neuronal PrPC (PARKYN et al., “LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein,” J Cell Sci., 2008, Vol. 121(Pt 6), pp. 773-83, Epub., 2008 Feb. 19). The function of the normal PrPC is not known, but there is evidence that it may function as a copper-dependent antioxidant, a signaling molecule, an anti- and pro-apoptotic molecule, as a protein supporting neuronal morphology and adhesion, and it may play a role in maintenance of long-term memory (ZOMOSA-SIGNORET et al., Physiological role of the cellular prion protein. Vet Res. 2008, Vol. 39:9. Epub., 2007 Nov. 27. Review). It has been recently proposed that PrPC is a marker of long-term bone marrow hematopoetic stem cells and supports their self-renewal (ZHANG et al., “Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal,” Proc Natl Acad Sci, USA, 2006, Vol. 103, pp. 2184-9, Epub., 2006 Feb. 7).
No differences in the primary structure (i.e. amino acid sequence) of PrPC and PrPd have been detected, nor have any differences been found between PrP genes or mRNAs from normal and infected brains with respect to structure or copy number. The physical differences (such as three-dimensional configuration) between the two proteins are therefore attributed to post-translational chemical modification. However, familial prion disease can occur in families with a mutation in the PrP gene, and mice with PrP mutations develop prion disease despite controlled conditions where transmission is prevented (HSIAO et al., “Spontaneous neurodegeneration in transgenic mice with prion protein codon 101 proline—leucine substitution,” Ann N Y Acad Sci., 1991, Vol. 640, pp. 166-70). Many different mutations have been identified and it is hypothesized that the mutations somehow make PrPC more likely to change spontaneously into the abnormal PrPd form.
PrPd is able to convert normal PrPC proteins into the infectious isoform by changing their conformation, or shape; this, in turn, alters the way the proteins interconnect. Data from animal transmission studies has pointed to the existence of an unidentified factor, termed “protein X,” which may control the conversion process (TELLING et al., “Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein,” Cell, 1995, Vol. 83, pp. 79-90). Although the exact 3D structure of PrPd is not known, during the refolding of PrPC into PrPd, some of the normal α-helical protein structure is partially converted into β-sheet. Aggregations of these abnormal isoforms form highly structured amyloid fibers, which accumulate to form plaques consisting of tightly packed β-sheets. Unlike PrPC, this altered structure is extremely stable and accumulates in infected tissue. This stability means that prions are largely resistant to denaturation by chemical and physical agents, making disposal and containment of the particles difficult. The term “PrPres” (the “res” is from “resistant”) is generally used to refer to the resistant proteolytic cleavage product of PrPd after treatment with Proteinase K.
Prions cause neurodegenerative disease by damaging neurons within the central nervous system and disrupting the normal tissue structure. While the incubation period for prion diseases is generally quite long, once symptoms appear the disease progresses rapidly, leading to brain damage and death. All known prion diseases are currently untreatable and fatal. Many different mammalian species can be affected by prion diseases. Due to the minor differences in PrP between different species, it is not unusual for a prion disease to be transmitted from one species to another. However, species to species transmission can only occur under certain conditions, and mechanisms of the transmission are not fully understood. The most recent example of such transmission is variant Creutzfeldt-Jakob disease (vCJD) affecting humans, which is believed to be caused by a prion which typically infects cattle, causing bovine spongiform encephalopathy (BSE), that was transmitted through contaminated bovine-derived food products (WILL et al., “A new variant of Creutzfeldt-Jakob disease in the UK,” Lancet, 1996, Vol. 347, pp. 921-5).
The primary route of natural TSE infection, for example scrapie in sheep and goats, BSE in cattle and sheep, CWD in deer and elk, and vCJD in humans, is thought to be through ingestion of contaminated sources. Prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids (e.g. in the case of CWD) (HALEY et al., “Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay,” PLoS ONE, 2009, Vol. 4, p. 4848, Epub., 2009 Mar. 18). They may then linger in the soil by binding to clay and other minerals (SAUNDERS et al., Prions in the environment: occurrence, fate and mitigation. Prion. 2008 Vol. 2:162-9. Epub 2008 Oct. 26. Review.). Other methods of infection are also known.
Low density lipoprotein receptor-related protein associated protein 1, also known as LRPAP1 or Receptor-Associated Protein (RAP), is encoded in humans by the LRPAP1 gene (STRICKLAND et al., “Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen”. J. Biol. Chem., 1991 Vol. 266, pp. 13364-9. KORENBERG et al., “Chromosomal localization of human genes for the LDL receptor family member glycoprotein 330 (LRP2) and its associated protein RAP (LRPAP1)”. Genomics 1994, Vol. 22, pp. 88-93. The protein was first isolated from mice as a 44-kD heparin-binding protein and was initially termed HBP-44 (FURUKAWA et al., “A heparin binding protein whose expression increases during differentiation of embryonal carcinoma cells to parietal endoderm cells: cDNA cloning and sequence analysis,” J. Biochem., 1990, Vol. 108, No. 2, pp. 297-302). In humans a 39-kD associated protein was purified as a part of the alpha-2-macroglobulin receptor complex (ASHCOM et al., “The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin,” J. Cell. Biol., 1990, Vol. 110, pp. 1041-8; STRICKLAND et al., “Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen,” J Biol Chem., 1991, Vol. 266, pp. 13364-9. The primary structure of the 39-kD polypeptide, termed alpha-2-macroglobulin receptor-associated protein (α2MRAP), was determined by cDNA cloning (STRICKLAND et al., “Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen,” J Biol Chem., 1991, Vol. 266, pp. 13364-9). Functional studies revealed that RAP blocked ligand binding by LRP1 (HERZ et al., “39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor,” J. Biol. Chem., 1991, Vol. 266, pp. 21232-8; WILLIAMS et al., “A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein,” Biol Chem., 1992, Vol. 267, pp. 9035-40). The deduced amino acid sequence of human RAP contains a putative signal sequence that precedes the 323-residue mature protein. The sequence showed 73% identity with a rat protein and 77% identity to a 44-kD mouse HBP-44. There are also similarities between RAP and apolipoprotein E. RAP is localized in the rough endoplasmic reticulum where it binds to LDL-receptor related proteins functioning as a specialized chaperone assisting in the folding and intracellular transport of members of the LDL receptor family. RAP is expressed in various organs and tissues throughout the body, including the brain. Experimental evidence suggests that RAP acts as a receptor antagonist and prevents association of newly synthesized LDL-receptor related proteins with their ligands during transport to the cell surface (WILLNOW, “Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors,” Biol Chem., 1998, Vol. 379, pp. 1025-31). RAP is efficiently transferred across the blood-brain barrier and may provide a means of protein-based drug delivery to the brain (PAN et al., “Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier,” J Cell Sci., 2004, Vol. 117(Pt 21), pp. 5071-8, Epub., 2004 Sep. 21). Recently, the importance of RAP has been shown in amyloid depositions in a mouse model of Alzheimer's disease (XU et al. “Receptor-associated protein (RAP) plays a central role in modulating Abeta deposition in APP/PS 1 transgenic mice,” PLoS ONE, 2008, Vol. 3, p. 3159). RAP also inhibited beta-amyloid protein (Abeta)oligomerization, neurotoxic effects of Abeta in cell cultures and blocked an Abeta-induced inhibition of long-term memory consolidation in 1-day-old chicks (KERR et al., “Inhibition of Abeta aggregation and neurotoxicity by the 39-kDa receptor-associated protein”, J Neurochem. 2010 Vol. 112:1199-209. Epub 2009 Dec. 10).